RADICAL SAM ENZYMES IN THIENAMYCIN BIOSYNTHESIS
Embargo until
Date
2015-12-07
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Johns Hopkins University
Abstract
Despite its broad anti-infective activity, the biosynthesis of the paradigm carbapenem antibiotic, thienamycin, remains largely unknown. Apart from the first two biosynthetic steps shared with a simple carbapenem, the pathway sharply diverges to the more structurally complex members of this class of beta-lactam antibiotics like thienamycin. Existing evidence points to three putative cobalamin-dependent, radical S- adenosylmethionine (RS) enzymes, ThnK, ThnL and ThnP, potentially being responsible for assembly of the ethyl side chain at C6, bridgehead epimerization at C5, installation of the C2-thioether side chain and C2/3-desaturation. The C2 substituent has been demonstrated to be derived from stepwise truncation of coenzyme A, but the timing of these events with respect to C2—S bond formation is not known. Using in vitro reactions with synthetically-accessed substrates, we show that ThnK of the three apparent cobalamin-dependent RS enzymes performs sequential methylations to build out the C6- ethyl side chain in a stereocontrolled manner. This enzymatic reaction produced the expected RS methylase coproducts S-adenosylhomocysteine (SAH) and 5’- deoxyadenosine (5’-dA) by LC-MS and was also found to require cobalamin. For double methylation to occur, the carbapenam substrate must bear a coenzyme A-derived C2- thioether side chain, implying the activity of a prior sulfur insertion by an unidentified enzyme. Heterologous expression of thienamycin biosynthetic enzymes in Streptomyces combined with a competition bioassay now implicates ThnL in attachment of the thioether. Additional substrate profiling with ThnK suggests that CoA itself is added to the bicyclic carbapenam nucleus. These insights allow refinements of the central steps in complex carbapenem biosynthesis.
Description
Keywords
Thienamycin, Radical SAM, Carbapenems